Folates in Plants: Research Advances and Progress in Crop

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Folates in Plants: Research Advances and Progress inCrop Biofortification.

Vera Gorelova, Lars Ambach, Fabrice Rébeillé, Christophe Stove, Dominiquevan der Straeten

To cite this version:Vera Gorelova, Lars Ambach, Fabrice Rébeillé, Christophe Stove, Dominique van der Straeten. Folatesin Plants: Research Advances and Progress in Crop Biofortification.. Frontiers in Chemistry, FrontiersMedia, 2017, 5, pp.21. �10.3389/fchem.2017.00021�. �hal-01518082�

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REVIEWpublished: 29 March 2017

doi: 10.3389/fchem.2017.00021

Frontiers in Chemistry | www.frontiersin.org 1 March 2017 | Volume 5 | Article 21

Edited by:

Arnaud Bovy,

Wageningen University and Research

Centre, Netherlands

Reviewed by:

Amarendra Narayan Misra,

Central University of Jharkhand, India

Giovanna Frugis,

Consiglio Nazionale Delle Ricerche

(CNR), Italy

*Correspondence:

Dominique Van Der Straeten

[email protected]

Specialty section:

This article was submitted to

Agricultural Biological Chemistry,

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Frontiers in Chemistry

Received: 16 December 2016

Accepted: 09 March 2017

Published: 29 March 2017

Citation:

Gorelova V, Ambach L, Rébeillé F,

Stove C and Van Der Straeten D

(2017) Folates in Plants: Research

Advances and Progress in Crop

Biofortification. Front. Chem. 5:21.

doi: 10.3389/fchem.2017.00021

Folates in Plants: Research Advancesand Progress in Crop Biofortification

Vera Gorelova 1, Lars Ambach 2, Fabrice Rébeillé 3, Christophe Stove 2 and

Dominique Van Der Straeten 1*

1 Laboratory of Functional Plant Biology, Department of Biology, Ghent University, Ghent, Belgium, 2 Laboratory of Toxicology,

Department of Bioanalysis, Ghent University, Ghent, Belgium, 3 Laboratoire de Physiologie Cellulaire Végétale, Bioscience

and Biotechnologies Institute of Grenoble, CEA-Grenoble, Grenoble, France

Folates, also known as B9 vitamins, serve as donors and acceptors in one-carbon (C1)

transfer reactions. The latter are involved in synthesis of many important biomolecules,

such as amino acids, nucleic acids and vitamin B5. Folates also play a central role in the

methyl cycle that provides one-carbon groups for methylation reactions. The important

functions fulfilled by folates make them essential in all living organisms. Plants, being

able to synthesize folates de novo, serve as an excellent dietary source of folates for

animals that lack the respective biosynthetic pathway. Unfortunately, the most important

staple crops such as rice, potato and maize are rather poor sources of folates. Insufficient

folate consumption is known to cause severe developmental disorders in humans. Two

approaches are employed to fight folate deficiency: pharmacological supplementation

in the form of folate pills and biofortification of staple crops. As the former approach

is considered rather costly for the major part of the world population, biofortification of

staple crops is viewed as a decent alternative in the struggle against folate deficiency.

Therefore, strategies, challenges and recent progress of folate enhancement in plants

will be addressed in this review. Apart from the ever-growing need for the enhancement

of nutritional quality of crops, the world population faces climate change catastrophes

or environmental stresses, such as elevated temperatures, drought, salinity that severely

affect growth and productivity of crops. Due to immense diversity of their biochemical

functions, folates take part in virtually every aspect of plant physiology. Any disturbance to

the plant folate metabolism leads to severe growth inhibition and, as a consequence, to a

lower productivity. Whereas today’s knowledge of folate biochemistry can be considered

very profound, evidence on the physiological roles of folates in plants only starts to

emerge. In the current review we will discuss the implication of folates in various aspects

of plant physiology and development.

Keywords: folate, vitamin B9, biofortification, stress response, plant development, metabolism, methylation,

neural tube defects

INTRODUCTION

Folates are indispensable components of metabolism in all living organisms (Bekaert et al., 2008).They play a role of donors and acceptors of one-carbon groups in one-carbon transfer reactions thattake part in formation of numerous important biomolecules, such as nucleic acids, panthothenate(vitamin B5), amino acids. Supplying methyl groups for methyl cycle, folates are involved in

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Gorelova et al. Folates in Plants

methylation reactions that are not only of primary importancein regulation of gene expression, but are also necessary for thesynthesis of lipids, proteins, chlorophyll and lignin.

Folates are synthesized de novo in bacteria, fungi and plants.Like other vertebrates, humans fully depend on their dietfor folate supply. Being an important component of humandiet, plants constitute the main source of folates for humanpopulation. Unfortunately, most staple crops such as potato,rice, cassava and corn are relatively poor in folates; hence, inregions where these staples are the main (or sole) energy source,folate deficiency is highly prevalent (Blancquaert et al., 2014).Insufficient consumption of folates was reported to be causallylinked with various developmental defects and diseases, such as,neural tube defects and anemia. In addition, folate deficiencyhas been correlated with an increased risk for cardiovasculardiseases, dementia and certain cancers (Blancquaert et al.,2010). In order to prevent the occurrence of such malignantdisorders, enhancement of folate supplementation has beenundertaken. Improvement of the nutritional value of staple cropsis considered the most affordable and sustainable approach.Although significant success has already been achieved in theenhancement of folate content in a number of plant species, suchas rice, tomato and lettuce (de La Garza et al., 2007; Storozhenkoet al., 2007a; Nunes et al., 2009), some hindrances are to betackled on the way to folate-rich crops. Indeed, biofortificationstrategies proved not to be equally efficient for different plantspecies, presumably due to differences in the regulation of thebiosynthetic pathway. The inherent instability of the folate pool(Arcot and Shrestha, 2005; Quinlivan et al., 2006; De Brouweret al., 2007) is another problem that has to be solved in orderto prevent folate loss during post-harvest manipulations andstorage. Moreover, in order to prevent possible interference withcritical processes the effects of folate overproduction on overallplant metabolism have to be thoroughly studied.

Besides being essential for human health, folates are importantfor plant wellbeing as well. Thus, proper functioning of folatemetabolism was demonstrated to be indispensable for plantdevelopment (Gambonnet et al., 2001; Ishikawa et al., 2003;Jabrin et al., 2003; Mehrshahi et al., 2010). Folates were reportedto play important roles in signaling cascades (Stokes et al., 2013),as well as in nitrogen and carbon metabolism (Jiang et al., 2013;Meng et al., 2014). Folate supplementation was demonstratedto improve plant biotic stress resistance (Wittek et al., 2015).Moreover, folate metabolism was shown to be differentiallyregulated in response to various abiotic stress conditions (Baxteret al., 2007; Neilson et al., 2011), that pointed out its importanceand possible specific adjustment in response to different stresses.Altogether these findings indicate that physiological roles andregulation of folate metabolism during development and stressresponse are important elements to be considered in the pursuitof crops with better productivity and improved stress tolerance.

In this review various aspects of folates in plants willbe addressed, such as their chemical properties, biosynthesis,metabolic roles and turnover. Understanding of these aspects isa prerequisite to the development of a successful biofortificationstrategy. This paper also summarizes recent findings on the rolesof folate metabolism in plant development and its link with other

metabolic processes and signaling pathways, as well as points outthe role of folates in plant stress response.

CHEMISTRY OF FOLATES

The term “folates” is generic for tetrahydrofolate (THF) andits derivatives. THF molecule is composed of three moieties: apterin ring, a para-aminobenzoate (pABA) and a glutamate tail(Figure 1). Naturally occurring folates are dihydrofolate (DHF)and tetrahydrofolate (THF), which differ by the oxidation stateof the pterin ring. While THF is a biologically active ready-to-usefolate form, DHF requires reduction by dihydrofolate reductase(DHFR). A folate molecule with a fully oxidized pterin ring iscalled folic acid. Folic acid requires two rounds of reduction inorder to become biologically active. THF molecules carry one-carbon units of various oxidation states attached to their N5and/or N10 positions (Figure 1). The type of a one-carbon unitloaded onto a THF molecule determines its metabolic function.Folate molecules can also be distinguished by the number of

FIGURE 1 | Structure of THF and its derivatives. (A) Structure of THF

molecule. Red arrows indicate positions of one-carbon groups. R1 and R2

represent various one-carbon substituents. (B) Substituents carried by a THF

molecule.






FIGURE 2 | Folate biosynthesis and regulation in plants. Black arrows indicate biosynthetic steps, red arrows and blunt-end arrows show activation and

inhibition, respectively, of enzymatic steps by folate precursors. Precursors: GTP, guanosine triphosphate; DHN-P3, dihydroneopterin triphosphate; DHN-P,

dihydroneopterin monophosphate; DHN, dihydroneopterin; HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, 6-hydroxymethyldihydropterin pyrophosphate; DHP,

dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; THF-Glu(n), tetrahydrofolate polyglutamate; ADC, aminodeoxychorismate; pABA, para-aminobenzoic acid.

Enzymes: GTPCHI, GTP cyclohydrolase I; DHN-P3-diphosphatase, dihydroneopterin triphosphate pyrophosphatase; DHNA, dihydroneopterine aldolase; HPPK,

HMDHP pyrophosphokinase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate synthetase; ADCS, aminodeoxychorismate

synthase; ADCL, aminodeoxychorismate lyase; GGH, gamma-glutamyl hydrolase.

glutamate residues that varies from 4 to 6 in plant naturallyoccurring folates.

FOLATE BIOSYNTHESIS IN PLANTS

While animals entirely depend on their dietary sources forthe folate supply, plants, bacteria and fungi can synthesizefolates de novo. In plants, THF biosynthesis is carried outin 11 steps and localizes to three subcellular compartments(Figure 2). Pterin and pABA moieties of THF molecule aresynthesized in cytosol and plastids, respectively. Final five stepsof the biosynthetic pathway are found to be mitochondrial andresult in the production of polyglutamylated THF [THF-Glu(n)].The biosynthetic steps are well-conserved among organisms-producers of folates. In the review we describe the thoroughly-characterized folate biosynthesis pathway in plants and draw aparallel with that in other organisms.

Pterine Synthesis in CytosolGTPCHISynthesis of the pterine moiety starts with the conversionof GTP into dihydroneopterin triphosphate and formate, areaction catalyzed by GTP cyclohydrolase I (GTPCHI). Thisconversion represents a rate-limiting step controlling metabolicflux into the folate pathway (Yoneyama and Hatakeyama, 1998;Hossain et al., 2004). GTPCHI from E. coli, yeast and mammalshave been cloned and characterized (Nar et al., 1995; Nardeseet al., 1996; Auerbach et al., 2000). Although mammals lack denovo folate biosynthetic pathway, GTPCHI is present in theirgenome and participates in tetrahydrobiopterin synthesis. E. coliand mammalian GTPCHI exist in a form of homodecamerscomprising five tightly bound dimers. Studies on spinach,

tomato, Arabidopsis and barrelclover (Medicago truncatula)demonstrate that plant GTPCHI has two tandem GTPCHIdomains (Sohta et al., 1997). The tandem configuration of theproteins was shown to be an absolute requirement for theirenzymatic activity, since the separate domains failed to exhibitany GTPCHI activity (Basset et al., 2002). The plant enzymeswere shown to be cytosolic (Basset et al., 2002; McIntosh andHenry, 2008; McIntosh et al., 2008). The presence of GTPCHItranscript in developing seed tissues, leaves and roots in wheatsuggested that de novo folate synthesis can occur throughout aplant body (McIntosh and Henry, 2008; McIntosh et al., 2008).

Dephosphorylation of Dihydroneopterin TriphosphateNext, dihydroneopterine undergoes dephosphorylation thatproceeds in two steps. The first step that is common in plantsand bacteria is the removal of pyrophosphate by cytosolic Nudixhydrolase (Klaus et al., 2005b). The second dephosphorylationstep is carried out by a non-specific phosphatase (Suzuki andBrown, 1974).

DHNAThe final step of the synthesis of the pterin moiety is catalyzedby dihydroneopterine aldolase (DHNA), which cuts the lateralside chain of dihydroneopterin releasing glycolaldehyde and 6-hydroxymethyldihydropterin (HMDHP) (Goyer et al., 2004).Plant DHNA activity is supported by three isoforms (Goyeret al., 2004) which, like their bacterial counterparts, aremonofunctional DHNA enzymes (Güldener et al., 2004). In fungiand protozoa, DHNA activities were shown to be coupled withother enzymes of the folate synthesis pathway: with HPPK andDHPS in the former and with DHPS in the latter (Güldener et al.,






2004). Due to the lack of obvious targeting signals, plant DHNAwas defined as a cytosolic protein (Goyer et al., 2004).

pABA Synthesis in PlastidsIn plants, pABA is synthesized in pastids from chorismate in twosteps (Nichols et al., 1989).

ADCSFirst, chorismate and glutamine are converted toaminodeoxychorismate and glutamate in the reaction catalyzedby aminodeoxychorismate synthase (ADCS). Like its fungal(Edman et al., 1993; James et al., 2002) and protozoan (Trigliaand Cowman, 1999) counterparts, plant ADCS exists as abipartite protein with tandem domains homologous to PabA(the glutamine amidotransferase) and PabB subunits (theaminodeoxychorismate synthase) of E. coli ADCS (Bassetet al., 2004a,b). In plants, it was shown that the NH3 releasedfrom the glutamine by the first domain is channeled towardthe second domain to react with chorismate to form theaminodeoxychorismate (Camara et al., 2011).

ADCLSecond, aminodeoxychorismate is converted to pABA in thereaction mediated by aminodeoxychorismate lyase (ADCL)(Basset et al., 2004a,b). In E. coli, ADCL activity is supported bya monomeric PabC protein (Green et al., 1992), while in plantsa homodimeric ADCL enzyme has been characterized (Bassetet al., 2004a,b). Following its synthesis, pABA can be convertedto its glucose ester by the activity of UDP-glucosyltransferase(Quinlivan et al., 2003; Eudes et al., 2008a). The pABA esterhas no assigned function so far and is assumed to serveas a storage form of pABA. The esterification of pABA wasdemonstrated to occur at high rates in tomato fruits and leavesas well as in tissues of other plant species (Quinlivan et al.,2003). Assessment of the ADCL activity in various subcellularcompartments revealed its predominantly cytosolic localization(Quinlivan et al., 2003). Owing to its amphiphilic nature, pABAis a membrane permeable compound and therefore can be freelydistributed between subcellular compartments. Its esterificationin cytosol restricts such passive transport into compartments andestablishes a readily reclaimable storage form of pABA that canbe shuttled to mitochondria by a dedicated transporter to enterfolate biosynthesis (Quinlivan et al., 2003).

Both, ADCS and ADCL have been demonstrated to befeedback inhibited by high levels of pABA and its glucose ester, aswell as by some folate species (THF, 5-methyl-THF and 5-formyl-THF) (Basset et al., 2004a,b).

THF Synthesis in MitochondriaHPPK/DHPSThe synthesis of THF in mitochondria starts withpyrophosphorylation of HMDHP and its subsequentcoupling with pABA that results in the formation ofdihydropteroate. These two reactions are catalyzed by HMDHPpyrophosphokinase (HPPK) and dihydropteroate synthase(DHPS) enzymatic activities. In E. coli, HPPK and DHPS aremonofunctional enzymes (Dallas et al., 1992), while in plants

(Rébeillé et al., 1997), protozoa (Triglia and Cowman, 1994), andfungi (Güldener et al., 2004) these two enzymatic activities arecoupled on one protein. Fungal HPPK and DHPS are domains ofa trifunctional protein possessing also DHNA activity (Güldeneret al., 2004). In plants, DHPS is shown to be feedback inhibitedby DHP, DHF, and THF (Mouillon et al., 2002).

DHFSBy the next step of THF synthesis a glutamate residue is attachedto the carboxy part of the pABA moiety of dihydropteroate(DHP) to form dihydrofolate (DHF) in the reaction mediatedby dihydrofolate synthetase (DHFS). Plant DHFS, like its fungalhomolog, exists as a monofunctional enzyme. In bacteria, DHFSfunction is coupled with FPGS activity (Bognar et al., 1985).

DHFRThe penultimate step of the biosynthetic pathway is performedby dihydrofolate reductase (DHFR) and reduces DHF intoTHF. Like in protozoa, plant DHFR exists as a bifunctionalenzyme coupled with thymidylate synthase (TS) (Luo et al., 1993;Neuburger et al., 1996; Cox et al., 1999), or as a monofunctionalenzyme coupled with TS as a part of a multimeric complex(Toth et al., 1987). In bacteria, yeast and vertebrates, DHFRis a monofunctional enzyme. The enzyme was demonstratedto be localized in mitochondria and plastids in carrot and pea(Neuburger et al., 1996; Luo et al., 1997).

FPGSThe last step of the synthesis of folates is the attachment ofa glutamate tail to THF molecule in the reaction catalyzedby folylpolyglutamate synthetase (FPGS). While in bacteriaFPGS is coupled with DHFS enzyme, eukaryotes possessmonofunctional FPGS. In Arabidopsis, three isoforms of FPGSexist and are targeted to three subcellular compartments:mitochondria, cytosol and plastids (Ravanel et al., 2001), whichis in agreement with the presence of polyglutamylated folates inthese compartments (Neuburger et al., 1996; Chen et al., 1997;Orsomando et al., 2005). A study of Arabidopsis FPGS doublemutants, suggested that each individual isoform can localize tomultiple compartments (Mehrshahi et al., 2010). Mammalianand fungal FPGS proteins are encoded by genes that produceboth mitochondrial and cytosolic isoforms depending on thetranslation start site activated (Freemantle et al., 1995; DeSouzaet al., 2000). Polyglutamylation affects compartmentalization ofthe folate pool by increasing anionic nature of folate molecules(Appling, 1991). Moreover, polyglutamylated folates are favoredby folate-dependent enzymes over their monoglutamate forms(Shane, 1989). Hence, polyglutamylation can be regarded asan important regulatory point of the folate metabolism. FPGSactivity is affected by the folate availability in the cell: high folateabundance inhibits FPGS activity in plants (de La Garza et al.,2007; Storozhenko et al., 2007a) and in mammals (Tomsho et al.,2008), while depletion of folate pool by the application of a folatesynthesis inhibitor, methotrexate (MTX), results in an increase ofFPGS activity (Loizeau et al., 2008). The regulation is assumed tooccur on both transcript and protein levels (Loizeau et al., 2008).






FIGURE 3 | Folate breakdown and salvage in plants. Red arrowheads indicate bonds prone to oxidative cleavage. PTAR, pterin aldehyde reductase; GGH,

gamma-glutamyl hydrolase; PGH, pABA-Glu hydrolase; HMDHP, 6-hydroxymethyldihydropterin; pABA, para-aminobenzoic acid.

FOLATE SALVAGE

Plants are continuously exposed to various stresses that resultin elevation of reactive oxygen species (ROS) level—thecause of oxidative stress. Folates, being inherently unstableentities (Arcot and Shrestha, 2005; Quinlivan et al., 2006;De Brouwer et al., 2007), are extremely vulnerable tooxidative damage. Upon oxidation, DHF and THF undergoa non-enzymatic C9-N10 bond cleavage that yields a pterintetrahydro- and dihydropterin-6-aldehyde, respectively, andpara-aminobenzoylglutamate (pABAGlu) (Figure 3) (Gregory,1989). Tetrahydro- and dihydropterin-6-aldehyde can befurther converted to the fully oxidized aromatic form, pterin-6-aldehyde (Whiteley et al., 1968; Reed and Archer, 1980;Hanson and Roje, 2001). Non-enzymatic cleavage is assumedto be the main route of the folate break-down, althoughinvolvement of enzymes is not excluded (Scott, 1984; Suh et al.,2001).

Folates vary in their susceptibility to the cleavage. Thus,tetrahydrofolate and dihydrofolate are most vulnerable to

degradation, while 5-formyl-THF and folic acid are most stablefolate forms (Reed and Archer, 1980; Gregory, 1989).

Folate break-down rates are suggested to be high in plants.This notion is based on post-harvest studies of leaves and fruitsthat point to the rates of approximately 10% per day (Scott et al.,2000; Strålsjö et al., 2003).The same folate break-down rates of10%were observed in the study on Arabidopsis plantlets suppliedwith an inhibitor of folate biosynthesis, sulfanilamide (Prabhuet al., 1998). Studies onmammals demonstrated that folate break-down products are excreted in urine (Scott, 1984), whereas fate ofthose in plants remains unclear. Some pieces of evidence suggestthat the products can be re-used for folate synthesis. Thus,despite the high rate of folate break-down, pABAGlu and pterinmoieties do not massively accumulate, as was demonstrated inthe study on Arabidopsis and pea tissues (Orsomando et al.,2006). Moreover, in vivo and in vitro studies shown that folatebreak-down products are readily converted into folate precursors(Orsomando et al., 2006; Noiriel et al., 2007b).

Upon the breakage of the C9-N10 bond, pABAGlu/pABAGlunand pterine moieties are further recycled. The recycling of






pABAGlu/pABAGlun starts with an enzymatic cleavage of theGlu tail, if any present, mediated by gamma-glutamyl hydrolase(GGH) (Figure 3) (Akhtar et al., 2008, 2010). The enzyme islocated in vacuoles (Orsomando et al., 2005; Akhtar et al., 2008).Despite a very high GGH activity, polyglutamylated folates stillexist in vacuoles. Two scenarios can be suggested to resolvethis paradoxical co-existence. First, it is possible that folatepolyglutamates are stabilized by folate binding proteins (FBP),as it was shown to occur in mammals (Hutchinson et al., 2000;Jones and Nixon, 2002). Plausibility of this hypothesis wasdemonstrated by a recent study showing an enhancement offolate pool stability in rice grains by expression of mammalianfolate binding protein (FBP) (Blancquaert et al., 2015), although,the existence of plant FBP remains to be demonstrated. Second,polyglutamylated folates might be sequestered away from highlyactive GGH in vacuoles (Orsomando et al., 2005).

In the next step, pABA is released from the Glu by the activityof pABAGlu hydrolase (PGH). Although plant PGH genes havenot been identified yet, PGH activities were found in Arabidopsis,pea and tomato (Orsomando et al., 2006). Studies on Arabidopsisand pea detected PGH activity in mitochondria, cytosol andvacuoles and suggested existence of at least two isoforms of theenzyme (Bozzo et al., 2008), which is in agreement with thefinding of two isoforms of the enzyme in E. coli (Hussein et al.,1998).

The recycling of the pterin cleavage product, dihydropterin-6-aldehyde, is achieved through its reduction to HMDHP, thefolate precursor (Orsomando et al., 2006). The reduction wasshown to be performed by non-specific pterin aldehyde reductase(PTAR) (Noiriel et al., 2007a,b). In pea, PTAR activity was foundto localize mainly in cytosol (at most 1% of the total activityis detected in mitochondria) and to be contributed by severalisoforms (Noiriel et al., 2007a,b). One of the PTAR isoformswas found to be encoded by At1g10310 gene in Arabidopsis.The predominance of PTAR localization in cytosol suggeststhat the break-down of pterin products (pterin aldehydes) mustbe transported from plastids and mitochondria to cytosol, inorder to be recycled. It is possible that a dedicated transporterconducting this shuttling exists. Dihydropterin-6-aldehyde thatdid not fall under recycling can be subjected to further oxidationto pterin-6-aldehyde, the fully oxidized form of the aldehyde.Plants lack the capacity to recycle the fully oxidized pterinaldehyde (Noiriel et al., 2007a,b). To date, only Leishmania isknown to reduce the fully oxidized pterin aldehyde (Bello et al.,1994). The reduction is performed by PTR1 enzyme that convertspterin-6-aldehyde to tetrahydropterin-6-aldehyde through 7,8-dihydro- state in a two-stage NADPH-dependent reaction (Belloet al., 1994). The lack of PTR1 activity in plants implies that thefully oxidized pterin aldehyde cannot be recycled, moreover, itcan be further oxidized to pterin-6-carboxylate. The oxidationcan occur in both non-enzymatic and enzymatic ways (Noirielet al., 2007a,b).

FOLATE TRANSPORT

Folate metabolism is known to function in several subcellularcompartments. Several pieces of evidence suggested existenceof interorganellar shuttling of folates and their precursors.

First, THF synthesis includes import of precursors, pABA andpterin, from plastids and cytosol, respectively, into mitochondria.Second, although folates are synthesized in mitochondria, theyare generously distributed throughout the plant cell, therefore,export of folates from the compartment must exist. Third,mutants compromised in folate biosynthesis can be rescued byexogenous folate application. Finally, folate transporters werefound in other species.

Unlike pABA, which is known to penetrate intracellularmembranes by diffusion (Quinlivan et al., 2003), pterins haveto be transported into mitochondria in order to be used infolate synthesis. Although the ability of plants to take up andmetabolize pterins is well known (Orsomando et al., 2006; Noirielet al., 2007a,b), no plant pterin transporter has been identifiedto date. Only a transporter competent of shuttling biopterin intoLeishmania cells has been described (Lemley et al., 1999).

Two transporters that facilitate folate transport intochloroplasts have been identified. Although being first identifiedas the closest mammalian mitochondrial folate transporter,AtFOL1 protein, encoded by At5g66380, was found to localizeto the envelope of chloroplasts. It proved to function as afolate transporter in Chinese hamster ovary (CHO) cells andE. coli mutant background. Inactivation of At5g66380 affectedneither the chloroplastic folate pool nor plant growth, suggestingexistence of an alternative plastidial transporter (Bedhommeet al., 2005).

The alternative chloroplast-localized transporter encoded byAt2g32040 gene rescues E. coli mutants unable to produceor transport folates. The Arabidopsis transporter was shownto shuttle 5-formyl-THF, folic acid and two antifolates, MTXand aminopterin. Interestingly, only monoglutamylated forms offolates could be transported (Klaus et al., 2005a). The Arabidosisgenome contains 8 homologs of At2g32040 gene. However, noneof the homologs proved to be functional in E. coli and Lieshmaniafolate and pterin transport mutant background (Eudes et al.,2010). The homologs were shown to lack some well-conservedresidues, supposedly affecting folate binding ability. As only 5-formyl-THF transport was investigated in the study, it is stillpossible that the homologs transport other folate derivativesand might have diverse specificity for different folate species.Disruption of At2g32040 significantly enhanced total folatecontent of chloroplasts and lowered abundance of 5-formyl-THF, but did not affect plant growth and development, whichreinforced the notion of functional redundancy between the twoidentified plastidial transporters.

A significant fraction of cellular folate pool localizes tovacuoles of the plant cell (Orsomando et al., 2005). Vacuolarfolate transporters were identified in Arabidopsis and red beet.Vacuolar membrane localized multidrug resistance-associatedprotein (MRP) AtMRP1 in Arabidopsis and its counterpartfrom red beet were demonstrated to function as transportersof folate mono- and polyglutamates, as well as antifolates(Raichaudhuri et al., 2009). It was demonstrated that disruptionof AtMRP1 causes elevated sensitivity to MTX, resulting fromhampered sequestration of the toxic compound in vacuoles. Thecompetence of the transporter to shuttle polyglutamylated formsof folates does not conform to the notion that folate transportersare specific for monoglutamylated forms of folates (Suh et al.,






2001; Klaus et al., 2005a; Eudes et al., 2008b). Finding that MTXpolyglutamates are transported from cytoplasm to lysosomes inmammalian cells (Barrueco and Sirotnak, 1991) suggested thattransporters capable of shuttling polyglutamylated folates arenot unique to plant vacuoles, moreover they can be conservedthroughout evolution.

Transporters assisting the passage of folates into cytosol areknown in mammals (Kamen et al., 1988). Mammalian cells werealso reported to take upMTX using reduced folate carrier (RFC)-mediated transport (Goldman et al., 1968; Dixon et al., 1994). Thenotion that folates can be taken up by a plant cell is supportedby numerous studies, demonstrating that exogenously suppliedfolate, 5-formyl-THF, can rescue mutants deficient in folateproduction (Mehrshahi et al., 2010; Meng et al., 2014; Reyes-Hernandez et al., 2014). Moreover, the notion is substantiated bythe known ability of plant cells to take-up antifolate drugs such asMTX (Cella et al., 1983). Additionally, plant cells are capable oftaking up pterins and using them in folate synthesis (Orsomandoet al., 2006; Noiriel et al., 2007a,b).

Import of folates into mammalian mitochondria was found tobe conducted by the inner membrane mitochondrial transportprotein (MTP). MTP could reconstitute the accumulation offolates within the mitochondrial matrix in a CHO mutant cellline that was deficient in this process (McCarthy et al., 2004).Import of folates in mitochondria certainly exists in plants, since,as mentioned above, Arabidopsis mutants defective in folatebiosynthesis can be rescued by application of 5-formyl-THF. Tobe metabolized, the folate derivative has to be converted to 5,10-methenyl-THF by the action of 5-formyl-THF cyclohydrolase(FTHFC), which localizes exclusively in mitochondria in plantcells (Roje et al., 2002b).

FUNCTIONS OF FOLATES

One of the most important cellular functions of folates istheir implication in DNA synthesis. Thus, folate deficiency inanimal tissues with rapidly dividing cells results in ineffectiveDNA synthesis (Kim, 2003; Kim et al., 2009). Lowered folatelevels were also found associated with impaired nucleotideproduction in plants (Srivastava et al., 2011). Namely, two folatederivatives contribute to DNA synthesis: 10-formyl-THF and5,10-methylene-THF (Figure 4). 10-formyl-THF is involved inthe synthesis of purine nucleotides by donating its one-carbonunit to carbon atoms 2 and 8 of the purine ring (Rowe, 1984; Liuand Lynne Ward, 2010).This folate species is also used for theproduction of formyl-methionyl-tRNA (Staben and Rabinowitz,1984; Schnorr et al., 1994) in the reaction catalyzed bymethionyl-tRNA transformylase in mitochondria and chloroplasts (Cossins,1987). 5,10-methylene-THF is used as a C1 donor by thymidylatesynthase (TS) in the conversion of dUMP into dTMP thatoccurs mitochondria (Neuburger et al., 1996). The localization ofbifunctional DHFR-TS proteins in carrot chloroplasts suggeststhat the conversion might take place in plastids as well (Luoet al., 1997). In the reaction 5,10-methylene-THF is convertedinto DHF that is subsequently reduced back to THF by DHFRenzymatic activity. Besides its role in thymidylate synthesis,

5,10-methylene-THF also serves as a C1 donor in the productionof pantothenate (vitamin B5), a precursor of co-enzyme A,and in the conversion of glycine to serine, an importantreaction during photorespiration in plants (Douce et al., 2001).Two subcellular compartments are potentially competent fornucleotide synthesis, namely mitochondria and plastids, as theyboth contain enzymes involved in the nucleotide production,such as TS, phosphoribosylglycinamide formyltransferase(GART) and phosphoribosylaminoimidazolecarboxamideformyltransferase (AICART). However, the flux of one-carbonunits from folate metabolism toward nucleotide synthesis isassumed to occur primarily in plastids (Neuburger et al., 1996;Atkins et al., 1997; Luo et al., 1997). In agreement with this, C13tracer studies demonstrated that the 5,10-methylene-THF poolin mitochondria predominantly shuttles between the glycinedecarboxylase complex (GDC) and the mitochondrial serinehydroxymethyltransferase (mSHMT) in the interconversion ofGly and Ser, rather than participating in thymidylate synthesis(Prabhu et al., 1996; Mouillon et al., 1999).

5,10-methylene-THF can be reduced by MTHFR into 5-methyl-THF, that enters methyl cycle where the one-carbongroup of 5-methyl-THF is utilized by methionine synthase inthe conversion of homocysteine (Hcy) to methionine. Thisreaction was found to occur in plant mitochondria (Clandininand Cossins, 1974), in chloroplasts (Shah and Cossins, 1970)and in cytosol (Eichel et al., 1995). Methionine is further usedby SAM synthetase in the production of S-adenosyl-methionine(SAM), that is employed by methyltransferases for methylationof DNA, RNA, lipids, histones and other substrates (Crider et al.,2012). Upon donating its methyl group, SAM is converted toS-adenosyl-homocysteine (SAH). Being a potent inhibitor ofMTHFR (Jencks and Mathews, 1987; Roje et al., 2002a), SAMregulates the flux of methyl groups from the folate pathway intomethyl cycle. Thus, low SAM levels favor methionine productionand vice versa (Crider et al., 2012). The one-carbon flux fromfolate metabolism toward methionine production is assumedto occur predominantly in the cytosol (Isegawa et al., 1993).The cytosol is also considered to be the only compartmentcapable of SAM production, since SAM synthetase is presentexclusively therein (Hanson and Roje, 2001). It is thereforesuggested that methylation reactions in plastids are supported bythe transport of SAM from the cytosol (Ravanel et al., 2004)AsSAH produced during methylation reactions is a potent inhibitorof methyltransferases (Moffatt and Weretilnyk, 2001), it has tobe efficiently eliminated. The removal of SAH is implemented bySAH hydrolase that recycles of SAH into SAM and is assumed tobe restricted to cytosol (Hanson and Roje, 2001). Export of SAHfrom chloroplasts was shown to be facilitated by a transporterthat imports SAM from the cytosol (Ravanel et al., 2004). Besidesbeing involved in methylation reactions, methionine can be usedin protein synthesis.

Exclusively in plants, THF is involved in photorespirationthat resides in three subcellular compartments: plastids,mitochondria and peroxisomes. During photorespiration,glycine is made from glycolate-2-P and is further convertedto 5,10-methylene-THF and CO2 by GDC. 5,10-methylene-THF is then combined with a second glycine by mSHMT to






FIGURE 4 | Metabolic functions of folates. THF, tetrahydrofolate; 5-CH3-THF, 5-methyltetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate;

5,10-CH+-THF, 5,10-methenyltetrahydrofolate; 5-CHO-THF, 5-formyltetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate; SAM, S-adenosyl-methionine; SAH,

S-adenosyl-homocysteine; Hcy, homocysteine; Met, methionine; Ser, serine; Gly, glycine. 1, serine hydroxymethyl transferase; 2, glycine decarboxylase complex; 3,

5,10-methylenetetrahydrofolate dehydrogenase; 4, 5,10-methenyltetrahydrofolate cyclohydrolase; 5, 10-formyltetrahydrofolate synthetase; 6, 5-formyltetrahydrofolate

cyclohydrolase; 7, 10-formyltetrahydrofolte synthetase; 8, 5,10-methylenetetrahydrofolate reductase; 9, thymidylate synthase; 10, methionine synthase; 11,

S-adenosylmethionine synthetase; 12, methyltransferase; 13, SAH hydrolase. Bolded reactions are specific for/ prevalent in a given subcellular compartment.

make serine (Cossins, 2000; Douce et al., 2001). While serineserves as the major source of single-carbon groups throughthe reverse reaction of SHMT, folates play a role to transportone-carbon units. 5,10-methylene-THF establishes a centralhub in one-carbon metabolism. It can be directly used for theproduction of thymidylate or pantothenate or can be convertedto 5-methyl-THF that enters methyl cycle. Moreover, it can beused for 10-formyl-THF synthesis that is carried out throughtwo sequential reactions catalyzed by a bifunctional methylenetetrahydrofolate dehydrogenase/methenyl tetrahydrofolatecyclohydrolase (MTHFD/MTHFC). 10-formyl-THF can beutilized in purine synthesis or in formyl-methionine-tRNAproduction. Finally, 10-formyl-THF is converted to THF, theunloaded folate form, by the action of 10-formyl-THF synthetase(FTHFS). The reloading of THF with a one-carbon unit isachieved through a reaction performed by SHMT where THFreceives its one-carbon group from serine. Serine that is formedfrom glycine and 5,10-methylene-THF in mitochondria can betransported to the cytosol and plastids (Rébeillé et al., 1994),where it is converted into 5,10-methylene-THF by cytosolicand plastidial SHMT activities, respectively (Neuburger et al.,1996).

Another source of one-carbon units for folate metabolismis formate. Formate is converted to 10-formyl-THFin the reaction catalyzed by FTHFS. The presenceof the enzyme in cytosol, plastids and mitochondria(Shingles et al., 1984; Kirk et al., 1994, 1995; Neuburgeret al., 1996) suggests that formate indeed can be analternative source of one-carbon groups for the folatemetabolism.

DISTRIBUTION OF FOLATES

The total folate pool is mainly composed of five THFderivatives: 5-methyl-THF (45–65%), 5-formyl-THF and 10-formyl-THF (30–55%) and THF and 5,10-methylene-THF (10–15%) (Cossins, 2000; Loizeau et al., 2007; VanWilder et al., 2009).Studies on folate composition in pea leaves demonstrated thatfolate derivatives are not equally distributed among subcellularcompartments (Jabrin et al., 2003; Orsomando et al., 2005).Mitochondria, being the actual site of de novo folate synthesis,contain more folates than other subcellular compartments (40%),while cytosol holds 30% and plastids and vacuoles contain 20%and 10%, respectively, of the total cellular folate pool (Jabrin et al.,2003; Orsomando et al., 2005). Onemay expect that folate speciesare distributed within a cell according to their function and themetabolic role of the compartment they reside in.

Thus, mitochondrial folate pool is dominated by THF and itsformyl derivatives (Orsomando et al., 2005). THF is synthesizedin mitochondria and further either directly transported toother subcellular compartments, or first loaded with one-carbonunits and then transported from mitochondria 5-formyl-THF,although is not being used as a one-carbon donor, represents50% of the formyl pool of mitochondria (Orsomando et al.,2005). It presumably serves as a storage folate form, which canbe converted to folate derivatives that can be used in one-carbontransfer reactions. Another possible function of 5-formyl-THF inmitochondria is the regulation of folate-dependent enzymes. Forinstance, it is known to inhibit activity of mitochondrial SHMT,thereby tuning the rate of photorespiration (Roje et al., 2002b;Goyer et al., 2005).






The prevailing folate derivative in the cytosol is 5-methyl-THF(Chen et al., 1997) which is readily used for methionine synthesiscoupled with SAM production to support methylation reactions.The plastidial folate pool is dominated by 5-methyl-THF and 10-formyl-THF (Orsomando et al., 2005) which can be employed inmethionine production and purine synthesis, respectively. Bothprocesses were shown to occur in plastids (Ravanel et al., 2004;Zrenner et al., 2006).

The length of the polyglutamate tail is another factor knownto affect distribution of folate species within a cell. Polyglutamatetails ensure retention of folates in subcellular compartments(Appling, 1991). Moreover polyglutamylated folate forms arepreferred by folate-dependent enzymes and folate transportersover their monoglutamylated counterparts (Rébeillé et al., 1994).Interestingly, when polyglutamylated folates are associated withfolate-dependent enzymes they are less susceptible to oxidativedegradation than when they are free in solution (Rébeillé et al.,1994). Most of cellular folates are polyglutamates with 4–6glutamate residues (Besson et al., 1993; Cossins, 2000).

Besides being unequally distributed between subcellularcompartments, folates vary in their abundance in differenttissues. Numerous studies pointed out that folate biosynthesisis most active in dividing tissues, such as root tips of peaand maize (Cox et al., 1999; Jabrin et al., 2003) or activelygrowing Arabidopsis cell suspension cultures (Loizeau et al.,2007, 2008). High expression of folate biosynthesis genes wasfound in meristems, expanding cotyledons and developing carrotembryos (Albani et al., 2005). High transcript abundance of folatebiosynthesis genes and elevated folate levels were also detected inpea embryos (Jabrin et al., 2003). The elevated level of folates inthese tissues might reflect a high demand for one-carbon unitsfor the nucleotide synthesis during the S-phase, as the genome isdoubled. In agreement with this, the expression of ribonucleotidereductase, the enzyme involved in nucleotide synthesis, is alsofound to be induced at the G1/S transition (Chabouté et al., 1998).Thus, it is very likely that genes of the folate biosynthesis pathwayare also regulated in a cell-cycle dependent manner.

QUANTITATIVE ANALYSIS OF FOLATES INPLANT SAMPLES, A PREREQUISITE INBIOFORTIFICATION PROGRAMS

In order to control the success of biofortification efforts,appropriate analytical methods are required to determine folatecontent in engineered plants. In experiments with geneticallymodified plants, analysts are often provided with only smallamounts of sample material. Further challenges in folate analysisinclude the distinction between different folate species, theirinstability and the complexity of the sample matrices (DeBrouwer et al., 2008).

Several methods for different food matrices such as fruit,juice, vegetables, potatoes, eggs, milk, meat and cereals havebeen published (Pfeiffer et al., 1997; Rader et al., 1998; Koningset al., 2001; Ndaw et al., 2001; Freisleben et al., 2003a,b; Rychlik,2004; De Brouwer et al., 2008, 2010; Van Daele et al., 2014).During sample preparation, it is crucial to consider conditions

that support the stabilization of folates, since folates are light-sensitive as well as sensitive to oxidants, reducers, acids and bases(Fitzpatrick et al., 2012). Interconversion between different folatespecies is also possible, especially for formyl folates (Jägerstad andJastrebova, 2013). Therefore, samples should generally be storedat −80◦C, all sample manipulations should be carried out undersubdued light conditions and antioxidants should be employed insample preparation (Van Daele et al., 2014; Strandler et al., 2015).Ascorbic acid is commonly used as an antioxidant. However,ascorbic acid can form formaldehyde when heated, which can inturn cause interconversion of folates. For this reason, thiols suchas dithiothreitol (DTT) are also added to the extraction buffer tocapture formaldehyde (De Brouwer et al., 2008; Strandler et al.,2015). Lyophilization of samples can be used to express the folatecontent per dry-weight but does not have a positive effect onfolate stabilization (Stea et al., 2007; Goyer and Sweek, 2011; VanDaele et al., 2014). For an extensive review of factors affectingthe stability and interconversion of folates, the publication byStrandler et al. is recommended (Strandler et al., 2015).

For starchy matrices, such as potato and rice, and for milk, theuse of a tri-enzyme treatment consisting of amylase, protease andconjugase has proven beneficial (Martin et al., 1990; De Brouweret al., 2008; VanDaele et al., 2014). Amylase and protease are usedto degrade starchy and protein components of the sample matrix,respectively, while conjugase cleaves the folate polyglutamate tailto produce mono- or diglutamates, depending on the sourceof conjugase (Strandler et al., 2015). Alternatively, the additionof conjugase can be skipped if only free monoglutamates areto be determined. For the analysis of folates in potatoes, tri-enzyme treatment did not enhance extraction efficiency but didfacilitate sample handling through matrix degradation; hence itmay decrease potential variability when different tuber sectionsare analyzed (Van Daele et al., 2014). In contrast, tri-enzymetreatment is necessary in the analysis of rice for optimal folateextraction (De Brouwer et al., 2008). Boiling steps are also usedin the preparation of starchy samples to soften the sample matrixand to reduce enzymatic and oxidative degradation (Van Daeleet al., 2014). For leafy matrices such as Arabidopsis thaliana,enzymatic treatment, except with conjugase where desired, is notrequired.

Extraction after tri-enzyme treatment can be followed bysimple ultracentrifugation (De Brouwer et al., 2008, 2010;Van Daele et al., 2014). More laborious and complex samplepreparation protocols may involve solid phase extraction (SPE)or affinity-based purification involving folate-binding protein(Wilson and Horne, 1984; Konings et al., 2001; Hyun andTamura, 2005).

Different techniques for the determination of folates insample extracts have been reported. Historically, microbiologicalassays were the method of choice. However, these assays aretime-consuming and cannot distinguish between differentfolate species (Strandler et al., 2015). In order to achievethis distinction, chromatographic methods are required.While a number of different detectors such as UV(-DAD),fluorescence, electrochemical and mass spectrometric detectorshave been reported in conjunction with high performanceliquid chromatography (HPLC) (Pfeiffer et al., 1997; Bagley






FIGURE 5 | Flow chart for determination of folates in plant material.

and Selhub, 2000; Ndaw et al., 2001; Rychlik and Freisleben,2002; Doherty and Beecher, 2003; Freisleben et al., 2003a; Zhanget al., 2003; Nelson et al., 2004; Fazili et al., 2005; Garratt et al.,2005; Zhang et al., 2005; De Brouwer et al., 2007; Patring andJastrebova, 2007; Gutzeit et al., 2008), (Ultra-) HPLC coupled totandem mass spectrometry [(U)HPLC-MS/MS] has become thede facto standard for the sensitive and selective determinationof folates (De Brouwer et al., 2010). UHPLC-MS/MS has alsobeen employed for the two-dimensional characterization offolate content in potato tubers (Van Daele et al., 2014). Forquantitative methods using mass spectrometric detection, theuse of isotope-labeled internal standards is essential in order tocompensate for both analyte loss during sample preparation andfor matrix effects (De Brouwer et al., 2008, 2010; Van Daele et al.,2014). Additionally, the use of a stable isotope dilution assay forthe determination of folates has been reported (Rychlik, 2004;Ringling and Rychlik, 2013). A flow chart outlining the generalanalytical steps for the determination of folates in plant materialis shown in Figure 5.

BIOFORTIFICATION

A large part of the world’s population is still suffering asevere deficiency of folate supply. To solve this problem, folatesupplementation in a form of folic acid pills was launched.Folic acid undergoes two rounds of reduction by DHFR to beconverted to the biologically active form, THF. Albeit beingrelatively simple and highly efficient, the approach turned outto be rather prohibitive in poor regions of the world, asthe pills often don’t reach the target populations. In additionto supplementation, industrial fortification of food products(e.g., flour) can be a possible approach to reduce folatedeficiency. However, the necessary infrastructure is often lackingin developing countries, especially in rural areas. An alternativeapproach to fight folate deficiency is the enhancement of folatelevels in staple crops. Both molecular breeding technologyand metabolic engineering can be used to create crops withenhanced micronutrient content, each with their advantagesand shortcomings (Blancquaert et al., 2017; Strobbe and VanDer Straeten, 2017). However, the first one is dependent onthe natural variation in germplasm of the crop of interest.Natural variation of folate content in rice (Abilgos Ramos, 2010;Blancquaert et al., 2010) and in potato (Goyer and Navarre,2007) was found to be relatively low, hampering successfulbreeding, keeping target levels in processed food in mind. Inseveral studies on the enhancement of folate levels in plants,GTPCHI or ADCS (or both), were overexpressed (Figure 6).The approach aims at the enhancement of the metabolic fluxthrough the biosynthetic pathway by increasing the supply ofthe two folate precursors—pterin and pABA. Overexpressionof a codon-optimized native GTPCHI in lettuce and E. coliGTPCHI in corn plants alone resulted in only 2.1–8.5-foldand 2-fold, respectively, increase in the folate level (Naqviet al., 2009; Nunes et al., 2009). The limited success of thetwo attempts was ascribed to the depletion of the pABApool. In agreement with this notion, a severe depletion ofpABA was observed in tomato plants overexpressing GTPCHI(Díaz de la Garza et al., 2004). In order to overcome thisobstacle, a simultaneous overexpression of GTPCHI and ADCSwas ventured. The approach successfully increased total folatecontent up to 25 and 100 times in studies on tomato andrice, respectively (de La Garza et al., 2007; Storozhenko et al.,2007a). Interestingly, the overexpression of ADCS alone lead to adecrease in the total folate content (Storozhenko et al., 2007a).It is possible that, being an inhibitor of FPGS (Storozhenkoet al., 2007a), pABA impedes folate polyglutamylation, thusaffecting folate stability and consequently its abundance. A studyon rice demonstrated that overexpression of HPPK-DHPS, agene coding for the first two enzymes of the mitochondrialpart of the biosynthetic path, results in a slight increaseof the folate level (Gillies et al., 2008). This result holdspromise for possibility of a further increase in folate level by acombined overexpression of GTPCHI, ADCS and HPPK-DHPSgenes.

Manipulation of the folate polyglutamylation status byaltering GGH expression was also demonstrated to affectfolate content. A study on tomato plants, reported that a






FIGURE 6 | Strategies of enhancement of folate level in plants. (A) Manipulation of GTPCHI expression, (B) manipulation of GTPCHI, ADCS and GGH

expression. Thickness of arrows indicates the efficiency of metabolic flux through the pathway. Precursors: GTP, guanosine triphosphate; DHN-P3, dihydroneopterin

triphosphate; DHN-P, dihydroneopterin monophosphate; DHN, dihydroneopterin; HMDHP, 6-hydroxymethyldihydropterin; HMDHP-P2, 6-hydroxymethyldihydropterin

pyrophosphate; DHP, dihydropteroate; DHF, dihydrofolate; THF, tetrahydrofolate; THF-Glu(n), tetrahydrofolate polyglutamate; ADC, aminodeoxychorismate; pABA,

para-aminobenzoic acid. Enzymes: GTPCHI, GTP cyclohydrolase I; DHN-P3-diphosphatase, dihydroneopterin triphosphate pyrophosphatase; DHNA,

dihydroneopterine aldolase; HPPK, HMDHP pyrophosphokinase; DHPS, dihydropteroate synthase; DHFR, dihydrofolate reductase; FPGS, folylpolyglutamate

synthetase; ADCS, aminodeoxychorismate synthase; ADCL, aminodeoxychorismate lyase; GGH, gamma-glutamyl hydrolase.

reduction of GGH expression by means of RNA interference,lead to an increase in polyglutamate tail and an increaseof the folate pool by 37%, whereas the overexpressionof GGH slashed folate content by 40% (Akhtar et al.,2010).

Due to the inherent instability of folate molecules,the total folate pool is prone to degradation upon fruitripening and storage. A recent study demonstrated thatthe expression of animal folate binding proteins increasesstability of folates upon storage (Blancquaert et al., 2015),probably because folates are less susceptible to oxidativedamage when associated with proteins (Rébeillé et al.,1994).

As future perspective for the enhancement of abundanceand stability of the folate pool in staple crops a combinationof several approaches should be ventured. It also shouldbe taken into account that biofortification strategies appliedmay not be equally efficient for all staple crop species.Thus, overexpression of both ADCS and GTPCHI substantiallyincreased folate level in tomato fruit (de La Garza et al.,2007) and rice grains (Storozhenko et al., 2007a), but didnot affect that of Arabidopsis and potato tubers (Blancquaertet al., 2013a). Another example of possible species-specificregulation of folate synthesis is shown by two studies onenhancement of pABA pool, where while the enhancement ofpABA pool did not affect total folate level in tomato plants(de La Garza et al., 2007), the increase of pABA level inrice resulted in a decrease of folate abundance (Storozhenkoet al., 2007a). Therefore, in order to develop a successfulbiofortification strategy, one should consider possible differencesin the regulation of folate biosynthesis of a given plantspecies.

ROLES OF FOLATES IN PLANTPHYSIOLOGY

Folates and Genome StabilitySince the genome encodes every developmental and metabolic

process in all living organisms, maintaining of its integrity is

of pivotal importance. Folates play a tremendously importantrole in maintaining genome stability (Fenech, 2001). Threefolate species are involved in this process: 5,10-methylene-THF, 10-formyl-THF and 5-methyl-THF. As was mentionedabove, 5,10-methylene-THF is utilized in the conversion ofdUMP into dTMP catalyzed by thymidylate synthase (TS).An insufficient conversion rate of dUMP to dTMP leads to adepletion of dTMP pool causing misincorporation of dUMP inDNA which ultimately affects genome stability. 10-formyl-THFis used for purine and formyl-methionyl-tRNA synthesis, thusbeing involved in nucleotide pool maintenance and translation.5-methyl-THF provides its methyl group for the production ofmethionine that is further used in the formation of the mostimportant methyl group donor for methylation reactions—SAM.Upon donating its methyl group, SAM is converted into S-adenosyl-homocysteine (SAH). Since SAH is a potent inhibitorof methytransferases, its removal is essential for the efficiencyof methylation reactions (Molloy, 2012). The importance ofmethylation reactions in the maintenance and regulation ofgenomes (epigenetic control) receives increasing attention. Asfolates play a role of methyl group donors, a positive correlationbetween the folate level and DNA methylation status seemsrather intuitive. However, numerous studies demonstrate thatthis scenario is not always realized. As was demonstratedin animal systems, folate deficiency can lead to global DNAhypomethylation (Balaghi and Wagner, 1993), to global DNA






hypomethylation with hypermethylation at specific genomeregions (Jhaveri et al., 2001; Pogribny and James, 2002), or toglobal DNA hypermethylation (Song et al., 2000; Sohn et al.,2003). Unlike animal systems, the role of folate metabolism inplant epigenome maintenance only starts to draw attention ofresearchers. So far, only examples of positive correlation betweenthe folate level and DNA methylation have been reported. Thus,Arabidopsis plants depleted in folates by the treatment with thefolate biosynthesis inhibitor sulfamethazine were shown to havereduced levels of DNAmethylation, histone H3K9 dimethylationand to exhibit release of epigenetic silencing (Zhang et al.,2012). The same study demonstrated a release of transcriptionalgene silencing in plants treated with methotrexate (MTX).Furthermore, disruption of the plastidial isoform of FPGS inArabidopsis was reported to result in reduced DNA methylationand release of chromatin silencing at a genome-wide scale (Zhouet al., 2013).

Modification of histones establishes another important aspectof epigenome. Like other histone marks, methylation of histonescan cause both transcriptional activation or silencing (Spenceret al., 1997; Xu et al., 1999; Lee et al., 2006). Recent studiesdemonstrate that transcription factors can also be subjectedto methylation, that can alter their DNA binding and proteinbinding efficiency (Salbaum and Kappen, 2011).

Since methylation of DNA, histones and transcription factorsis of paramount importance in regulation of gene expression, theinvolvement of folate metabolism in methylation reactions linksit to virtually every process in a cell.

Folates in Plant DevelopmentDue to their crucial role in methylation reactions and in theoverall cellular metabolism, folates are absolutely essential fordevelopment. The importance of folates for plant developmentwas demonstrated by several studies reporting a severe repressionof growth caused by inhibition of folate biosynthesis byapplication of antifolates (Crosti, 1981; Camara et al., 2012;Navarrete et al., 2013).

Several lines of evidence indicate that folates are indispensableduring embryogenesis. Thus, fpgs1/fpgs2 and dhfs (gla1) mutantscompromised in folate biosynthesis are embryo lethal (Ishikawaet al., 2003; Mehrshahi et al., 2010). Moreover, the folate levelin pea embryos was shown to be high (Gambonnet et al., 2001;Jabrin et al., 2003) and accompanied by high expression ofgenes involved in folate biosynthesis (Jabrin et al., 2003). Thisevidence suggested that plant embryos are autonomous for folatesynthesis. Application of inhibitors of folate synthesis indicatedthat embryos not only produce folates necessary for their ownproper development but also procure folates that are usedduring early postembryonic development (Gambonnet et al.,2001). As was shown in the experiment using antifolates, folatessupply accumulated during embryonic development is sufficientfor supporting cell division only during early post-germinationgrowth and it soon becomes a limiting factor (Gambonnet et al.,2001). Therefore, folate biosynthesis is assumed to resume shortlyafter germination. During organ differentiation folate synthesiswas found to be prevalent in highly dividing tissues, such as peaand maize root tips, maize kernels as well as in actively growing

cell suspension cells of carrot (Cox et al., 1999; Jabrin et al., 2003;Albani et al., 2005).

The crucial role of folates in root development wasdemonstrated by recent studies using Arabidopsis plants mutantfor the plastidial isoform of FPGS (AtDFB). While mutantsfor other FPGS isoforms did not exhibit obvious primary rootdefects, atdfb seedlings had a severely shortened primary root(Srivastava et al., 2011; Reyes-Hernandez et al., 2014). Thispointed to specificity of the plastidial isoform in maintainingC1 metabolism during root development. Although the totalfolate content in atdfb seedlings was not altered, folate speciesdistribution was disturbed. Mutant seedlings were also shownto be depleted in amino acids and nucleotides. The lowerednucleotide content was in agreement with significantly decreasedlevel of 10-formyl-THF which is used for the synthesis of purines.Furthermore, the mutants exhibited a lowered methionine level(Srivastava et al., 2011) and decreased abundance of 5-methyl-THF which donates its methyl group to homocysteine inmethionine synthesis (Ravanel et al., 2004; Rébeillé et al., 2006).Apparently, the plastidial isoform of FPGS is indispensable forsyntheses of purine and methionine that are known to take placein plastids.

The primary root defect of atdfb plants was attributed toerroneous regulation of the root determinacy-to-indeterminacyswitch (IDS) which is accompanied by the activation ofthe quiescent center (QC) and subsequent consumption ofthe root apical meristem (RAM). The activation of theQC was demonstrated to be dependent neither on auxingradients nor on SHORTROOT/SCARECROW (SHR/SCR) andPLETHORA (PLT) pathways of RAM indeterminacy (Reyes-Hernandez et al., 2014). Taking into account the pivotal roleof auxin in root development, the IDS independence of auxingradients demonstrated by the mutants seemed quite striking.Nevertheless, the result is supported by a study showing a similarlack of a direct link with auxin, reporting that RAM exhaustionand root determinacy of triple mutant of GRAS TF HAIRYMERISTEM 1,2,3 is independent of auxin gradients (Engstromet al., 2011).

Some pieces of evidence suggest that folates implement animportant role in hypocotyl development. Thus, a crosstalkbetween folate and sucrose was shown to influence auxinsignaling through a subset of IAA/ARFs to impact/regulatehypocotyl elongation (Stokes et al., 2013). Moreover, a crosstalkbetween folates and sucrose was previously observed in a studywhere seedling lethality of fpgs2 fpgs3 double mutants could bepartially rescued by supplementation with sucrose (Mehrshahiet al., 2010). Another example of the involvement of folatemetabolism in hypocotyl elongation is presented in a studyrevealing compromised hypocotyl elongation in atdfb mutantsduring skotomorphogenesis (Meng et al., 2014).

Folates and LightFolate synthesis and accumulation were found to be elevated inleaves upon exposure to light (Jabrin et al., 2003). This findingpointed out a high demand for folates and thus one-carbontransfer reactions in metabolism of leaf photosynthetic tissues.This notion was corroborated by the study demonstrating a






decrease in both total folate abundance and chlorophyll levelin pea leaves upon application of an antifolate drug MTX (VanWilder et al., 2009). The folate role in the methyl cycle wassuggested to be accountable for the link between chlorophyll andfolate levels. Thus, SAM, which synthesis involves 5-methyl-THF,is used bymethytransferases that methylate numerous substrates,including a precursor of chlorophyll (Von Wettstein et al., 1995;Suzuki et al., 1997). Moreover, some chloroplastic enzymes suchas Rubisco are also methylated (Black et al., 1987). Therefore,it is assumed that a high level of folates in leaves is neededfor the proper functioning of the photosynthetic apparatus.Beside their role in chlorophyll synthesis, folates establish anadditional link with plant light response. Folate molecules wereshown to bind cryptochromes cry1 and cry2 and act as light-harvesting antenna pigment, thereby participating in blue-lightperception (Hoang et al., 2008). It has been demonstratedthat in purified preparations of CRY-DASH-type cryptochromesof Vibrio cholera folate as a light-sensing antenna pigmentis involved in energy transfer to flavin which is the secondchromophore that binds to cryptochromes (Saxena et al., 2005).It is possible that a similar mechanism of blue-light perceptionoperates in plants. The enhancement of folate biosynthesis uponlight exposure could also be attributed to the role of folates inthe Gly-to-Ser transition during photorespiration. Upon lightexposure the expression of HPPK-DHPS was demonstrated tofollow the accumulation of transcripts of GDC and SHMT thatlink folate metabolism to photorespiration (Jabrin et al., 2003).

Folates and Nitrogen ReservesNitrogen is an essential macronutrient and one of the majorlimiting factors for plant growth (Diaz et al., 2006). Insufficientnitrogen supplementation results in perturbations of plantmetabolism and severe growth defects (Martin et al., 2002;Diaz et al., 2008; Lemaître et al., 2008). Thus, Arabidopsisseedlings mutant for either plastidial or mitochondrial FPGSdemonstrate erroneous folate species distribution and display adisturbed nitrogen metabolism within a specific developmentalwindow (Jiang et al., 2013; Meng et al., 2014). Mutants forthe plastidial form of FPGS were shown to be defective inseed reserves exhibiting a defective C and N partitioningcapacity (Meng et al., 2014). Plants lacking the mitochondrialFPGS isoform demonstrated root shortening upon nitrogen-limited conditions (Jiang et al., 2013). Both mutants exhibited atypical phenotype of plants under low-nitrogen stress, showinglowered soluble protein content, decreased free amino acidsabundance, low nitrate content and decreased abundance ofnitrogen storage amino acids and accumulation of NH+

4 .Since non-photorespiratory conditions could partially rescuethe phenotype of plants lacking mitochondrial FPGS under N-limited conditions, the growth defects were assumed to be in partcaused by impaired photorespiration (Jiang et al., 2013).

Folates in Stress ResponseWith a future of climatic instability, production of stress resistantcrops will become the major challenge in the near future.Deeper understanding of metabolic responses and signalingwould help to identify best candidates for genetic manipulations

for enhancement of crop resistance and productivity. Since plantstress responses include adjustment of a galore of metabolicprocesses and folates take part in a good deal of them, a tightand elaborate regulation of folate biosynthesis and metabolism inresponse to various stress conditions can be expected. However,the role of folates in plant stress response and resistancewas largely overlooked. Studies conducting transcriptome andmetabolome analyses provided evidence that folate metabolismis dramatically and differentially affected by various stressconditions. On the one hand, a study on Arabidopsis suspensioncell cultures reported an up-regulation of genes involved in folatebiosynthesis and consumption in response to oxidative stressimposed by application of menadione (Baxter et al., 2007). Onthe other hand, a global protein expression study investigatingthe proteomic response of rice plants to cold stress, demonstrateda down-regulation of folate biosynthesis (Neilson et al., 2011). Adecrease of folate biosynthesis related genes was also observed inplants under salt stress (Storozhenko et al., 2007b).

A link between folate depletion and oxidative stress in animalswas demonstrated by numerous studies (Cano et al., 2001; Huanget al., 2001; Ho et al., 2003; Dhitavat et al., 2005). In general,oxidative stress is caused by accumulation of reactive oxygenspecies (ROS) that include free radicals such as superoxide anion(O−

2 ), hydroxyl radical (HO◦), as well as non-radical moleculesincluding hydrogen peroxide (H2O2) and singlet oxygen. Inplants, ROS are largely formed by the leakage of electronson O2 from the electron transport activities of chloroplast,mitochondria and plasma membranes or as byproducts ofvarious processes from different compartments (Foyer andHarbinson, 1994; del Río et al., 2006; Blokhina and Fagerstedt,2010; Heyno et al., 2011). High levels of ROS are very harmful toan organism and therefore need to be neutralized by the cellularantioxidant system. The antioxidant system comprises enzymaticand non-enzymatic antioxidants. Superoxide dismutase (SOD),catalase (CAT), thioredoxin (TXN), and guaiacol peroxidase(GPX) establish enzymatic defense against ROS (Noctor andFoyer, 1998) while glutathione (GSH) and ascorbate (AsA), themain components of Asada-Halliway pathway, are the majorcellular redox buffers that constitute the non-enzymatic ROSdetoxifying system.

Besides being used in reductive biosynthetic processesinvolving those of nucleotide and fatty acid production, NADPHis used by the glutathione-ascorbate ROS detoxifying pathwayas a reducing agent for glutathione recycling. The cofactoris also employed by the enzymatic antioxidant thioredoxin.The major sources of NADPH production in plants are thephotosynthetic electron transfer chain in plastids (Kramer andEvans, 2011) and the oxidative pentose phosphate pathway,that takes place in the cytosol and in plastids (Kruger andvon Schaewen, 2003). Mitochondria, as the actual (major) siteof folate biosynthesis (Neuburger et al., 1996), contribute to alesser extent to NADPH accumulation. Reduction of NADP+

is attributed to a number of mitochondrial enzymes [isocitratedehydrogenase (EC 1.1.1.42), malic enzyme (EC 1.1.1.39), delta-pyrroline-5- carboxylate dehydrogenase (EC 1.5.1.12), glutamatedehydrogenase (EC 1.4.1.3) and methylenetetrahydrofolatedehydrogenase (EC 1.5.1.5) (Rasmusson and Møller, 1990;






FIGURE 7 | Scheme reflecting the role of folate metabolism in redox homeostasis in plants. The folate pathway possibly contributes to production of NADPH

that is used to detoxify ROS. Additionally, 5-CH3-THF takes part in the conversion of Hcy into Met, thereby preventing ROS production. Green areas represent

pathways contributing to NADPH production, blue areas show pathways consuming NADPH, biosynthetic processes and ROS removal. THF, tetrahydrofolate;

5-CH3-THF, 5-methyltetrahydrofolate; 5,10-CH2-THF, 5,10-methylenetetrahydrofolate; 10-CHO-THF, 10-formyltetrahydrofolate.

Møller and Rasmusson, 1998)]. The actual contribution of each ofthe mentioned sources to the total NADPH production in plantsremains unclear to date.

A recent NADP+ labeling study demonstrated that folatemetabolism contributes to the total NADPH production inanimals. The reaction that results in NADPH productionconverts 5,10-methylene-THF into 10-formyl-THF and isperformed by MTHFD (Fan et al., 2014) (Figure 7). The studydemonstrated that metastatic tumor cells exhibit an elevationin the production of NADPH, associated with increased activityof the folate pathway. This suggests that tumor cells evolvedan adaptation to buffer oxidative stress by activating the folatepathway (Fan et al., 2014).

The relationship of folate metabolism with oxidative stressdoes not seem to be restricted by the production of NADPH,though. Several studies suggest that folate pathway might belinked with ROS metabolism through homocysteine. Thus, anincrease of homocysteine level caused by low folate intake inhumans (Jacob et al., 1994; Bostom et al., 1996; Shimakawaet al., 1997) was found to exert toxicity on endothelial cells byincreasing H2O2 production (Starkebaum and Harlan, 1986),affecting antioxidant defense systems (Blundell et al., 1996).Being in agreement with the data, another study demonstratedthat culturing embryonic cortical neurons and differentiated SH-SY-5Y human neuroblastoma cells in folate-freemedium inducedneurodegenerative changes that were accompanied by increasedROS level resulted from elevation of homocysteine abundance(Ho et al., 2003).

Since folate metabolism is an essential part of basiccellular metabolism, one can expect a great conservation in

its functioning, roles and regulation throughout evolution.Therefore, it is possible that the role of the folate pathway inROS metabolism revealed in animal systems can be also foundin plants.

Several lines of evidence associate folates with plantinnate immunity. Thus, expression of defense-relatedgenes is induced in rice seeds with elevated folate level(Blancquaert et al., 2013b). Furthermore, application ofpABA induced systemic acquired resistance (SAR) againstartificially infiltrated Xanthomonas axonopodis and naturallyoccurring tobacco mosaic virus in pepper seedlings (Songet al., 2013). The importance of folate metabolism in bioticstress resistance was also demonstrated in a study assessingglobal gene expression patterns in response to the fungalpathogen Fusarium pseudograminearum (Powell et al., 2016).Finally, folate precursor DHN and folic acid induce localand systemic SA-mediated defense in Arabidopsis (Witteket al., 2015). Altogether, these findings suggest that folatemetabolism might have an important role in plant biotic stressresponse.

CONCLUSIONS

Until recently, plant folate field was dominated by biofortificationstudies. Although a significant amount of knowledge regardingfolate biochemistry and biosynthesis in various plant specieshas been accumulated, roles of folates in plant physiology werelargely overlooked. The implication of folates in many essentialprocesses such as synthesis of DNA and amino acids, methylationreactions suggests that these compounds might affect virtually






every process in plants. Recent findings provided compellingevidence for involvement of folates in various aspects of plantphysiology such as development, light response, nitrogen andcarbon metabolism and stress response. Perturbations to anyof the listed aspects lead to impaired plant growth and loss ofproductivity and ultimately affects agricultural value of crops.Although very scarce, the evidence on the role of folates in stressresponse holds promise for improvement of plant performancein unfavorable conditions. Study of physiological roles of folateswould not only deepen our understanding of functions of thesecompounds in plants but also has a potential to fill gaps in studiesof various processes in a plant cell.

AUTHOR CONTRIBUTIONS

VG conducted the literature search, drafted the manuscript andprepared figures. LA drafted the part on folate analysis andprepared Figure 5. FR, CS and DV helped in writing the finalmanuscript.

ACKNOWLEDGMENTS

DV acknowledge support from Ghent University (BijzonderOnderzoeksfonds BOF2009/G0A/004), and the ResearchFoundation – Flanders (FWO, projects 3G012609 and 35963).

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